Project IV- Substrate Docking in Cytochrome c Oxidase (Ferguson-Miller, Kuhn, Garavito, Roberts) Cytochrome c oxidase is an intrinsic membrane protein whose complex energy transducing function involves electron transfer, reduction of oxygen to water and translocation of protons across the membrane. This proposal is aimed at determining the nature of the protein-protein interaction between cytochrome c and cytochrome c oxidase, which determines the rate and efficiency of electron delivery in the oxidase. Models will be developed for the docking sties for cytochrome c on CcOX by a powerful computational algorithm that uses protein electrostatic fields and van der Waals surfaces in a systematic orientation search of the intermolecular energies of the two proteins. The predictions of this model will be tested by mutation of Rhodobacter sphaeroides CcOX, comparison of the kinetics and binding of cytochrome c with the mutant and wild-type enzymes, and by crystallizing native and mutant forms of Rhodobacter CcOX and CcOX/cytochrome c complexes, to determine their structure by X-ray analysis. Homology modeling of the Rhodobacter sequence into the bovine oxidase coordinates (available to us from our collaborator, S. Yoshikawa) will permit computational analysis of the docking with a model closer to that of the bacterial enzyme. Conversely, creation of a chimeric bacterial enzyme with a mammalian coxII gene substituted for the Rhodobacter gene, will allow assays of binding to be done in a system that better corresponds to the mammalian oxidase structure and better matches the mammalian cytochrome c. Surface water at the oxidase/cytochrome c interface will also be analyzed to predict retained water molecules that may influence the chemistry of the interaction. Defining the role of electrostatics and other forces in this protein-protein interaction will increase our understanding of the mechanism and mechanics of electron transfer in the respiratory chain.